Chromatin Immunoprecipitation and Quantitative PCR.

ChIP assay was performed by using standard procedure. Proteins were cross-linked to genomic DNA with 1% formaldehyde for 10 min at 23 °C. The cross-link was stopped by 0.125 M glycine for 5 min at 23 °C. The cells were washed three times with ice-cold PBS and lysed in 0.45 mL of RIPA buffer with proteinase inhibitor mixtures on ice for 10 min. Cell pellet was sonicated five cycles by using Bioruptor XL (8 min/cycle, 30 s on and 30 s off) and then spun down at 14,000 × g for 10 min to remove debris. The lysates were precleared by incubating with a salmon sperm DNA/protein A agarose slurry for 30 min at 4 °C with rotation. The cleared lysates were diluted 1:6 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris⋅HCl, pH 8.0, and 167 mM NaCl). The samples were incubated with 1 μg of antibody for 18 h at 4 °C with rotation. Protein A agarose slurry (50 µL) were added and incubated for 2 h at 4 °C. The beads were washed once each with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris⋅HCl pH 8.0, 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris⋅HCl pH 8.0, 500 mM NaCl), LiCl buffer (250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris⋅HCl pH 8.0), and TE buffer (10 mM Tris⋅HCl pH 8.0, 1 mM EDTA). The beads were eluted twice with a total of 200 μL of elution buffer (1% SDS, 100 mM NaHCO₃) at 25 °C for 15 min with shaking (1,000 rpm in an orbital mixer). NaCl was added to 200 mM, and the samples were decross-linked at 65 °C for 18 h. Proteinase K (0.1 mg/mL), EDTA (10 mM), and Tris⋅HCl pH 6.8 (40 mM) were added and incubated at 42 °C for 1 h. DNA was extracted by using GenElute PCR clean-up kit (Sigma). The samples were subjected to SYBR Green real-time PCR analysis by using the following primers: MDM2 promoter (5′-CGG GAG TTC AGG GTA AAG GT and 5′-CCT TTT ACT GCA GTT TCG). p21 promoter (5′-TGG CTC TGA TTG GCT TTC TG and 5′-TTC AGA GTA AGA GGC TAA GG). PUMA promoter (5′-CTG TGG CCT TGT GTC TGT GAG TAC and 5′-CCT AGC CCA AGG CAA GGA GGA C).